DNA encoding CAI resistance proteins and uses thereof

ABSTRACT

This invention provides for nucleotide sequences that encode CAIR proteins correlated with cellular resistance to carboxyamido-triazole (CAI) and functionally equivalent compounds. The invention further provides for methods of detecting CAI resistance in biological samples and for cell lines that grow and proliferate in the presence of CAI and functionally equivalent compounds.

This is a Division of application Ser. No. 08/212,190, filed May 23,1994, U.S. Pat. No. 5,652,223, the disclosure of which is incorporatedby reference.

FIELD OF THE INVENTION

The present invention relates to the isolation of a CAI resistance(CAIR) gene that encodes a protein conferring cellular resistance tocarboxyamido-triazole (CAI) and functionally equivalent compounds. Thisinvention further relates to the CAIR protein encoded by the CAIresistance gene, antibodies specific to the protein, and nucleic acidprobes that specifically hybridize to the gene. Additionally theinvention provides assays for determining CAI resistance in cells, andassays to screen for compositions that obviate CAI resistance. Theinvention also provides for cell lines that express the CAIR gene andare capable of growing and proliferating in cultures chronically exposedto CAI and functionally equivalent compounds.

BACKGROUND OF THE INVENTION

Calcium homeostasis is important in the regulation of cellular behavioras it is paramount in regulation of signaling events as well as othercellular and molecular functions. (Cole and Kohn, Cancer and MetastasisRev. (1993). Carboxyamido-triazole (CAI) is an inhibitor of stimulatedcalcium influx through voltage gated and nonvoltage-gated calciuminflux. It has been shown to inhibit important downstream signalingevents including the release or arachidonic acid, production of inositoltriphosphate in response to phosphorylation and activation ofphospholipase A2, and tyrosine phosphorylation in response to receptoractivation (Felder, et al. J. Pharm. Exp. Therap., 257: 967-971 (1991);Gusovsky, et al. J. Bio. Chem., 268: 7768-7772 (1993)). These signallingpathways regulate proliferation and other cellular events includingadhesion, migration, and production of proteases. (Kohn, et al. CancerRes. (1994) in press, Kohn, et al. Proc. Natl. Acad. Sci. USA,communicated; Kohn and Liotta, J. Nat. Cancer Inst., 82: 54-60 (1990)).

Carboxyamido-triazole (CAI) has been observed to inhibit malignantproliferation, invasion, and metastasis of cancer cells, suggesting therole of CAI, and related compounds, as potential cancer therapeutics(see patent application U.S. Ser. No. 07/985,402now issued as U.S. Pat.No. 5,359,078). Of concern in the development and utilization of cancertherapeutics is the development of resistance by tumor cells to theparticular pharmacological regimen to which they are exposed.

Where such a resistance appears it is desirable to ascertain theresistance in order to devise therapeutics that obviate or prohibit theappearance of such resistance. When the resistance is associated withaltered gene expression, isolation of the gene encoding proteinsassociated with the appearance of the resistance not only aids in theelucidation of the mechanism of drug resistance, but also providesuseful markers for detecting the acquisition of the resistance as wellas useful targets for intervention. The present invention provides anisolated CAI resistance (CAIR) gene whose expression is correlated withCAI resistance. This invention also provides for the protein encoded bythis gene. The isolation of this gene and protein provide theaforementioned and other advantages.

SUMMARY OF THE INVENTION

The present invention provides for an isolated human nucleic acidencoding a human carboxyamido-triazole resistance (CAIR) protein wherethe nucleic acid is capable of hybridizing specifically to a secondnucleic acid consisting of the nucleic acid sequence of SEQ ID NO:1 inthe presence of a human genomic library under stringent conditions.Specifically this nucleic acid comprises the nucleotide sequence of SEQID NO:1. More particularly, the isolated nucleic acid, when found in acell line, expresses the protein it encodes at higher levels when thecell line is cultured in the presence of at least 10 μM concentration ofCAI than when the cell line is cultured under the same cultureconditions without CAI.

The invention also provides for an isolated human nucleic acid sequenceencoding a CAIR protein, wherein the nucleic acid sequence has at least85%, more particularly at least 95% sequence identity with the nucleicacid of SEQ ID NO:1. The isolated human nucleic acid sequence, whenfound in a cell line, expresses the protein it encodes at higher levelswhen the cell line is cultured in the presence of at least 10 μMconcentration of CAI than when the cell line is cultured under the sameculture conditions without CAI.

This invention additionally provides for an isolated human nucleic acidencoding a CAIR protein having at least 80%, more particularly at least95% amino acid identity with the amino acid sequence of SEQ ID NO:2.This isolated human nucleic acid, when found in a cell line, expressesthe protein it encodes at higher levels when the cell line is culturedin the presence of at least 10 μM concentration of CAI than when theline is cultured under the same culture conditions without CAI.

In another embodiment, this invention provides for an isolated CAIRprotein that has at least 80% sequence identity, more particularly atleast 95% sequence identity with the amino acid sequence of SEQ ID NO:2.This isolated CAIR protein, when expressed in a cell line, is expressedat higher levels when the cell line is cultured in the presence of atleast 10 μM concentration of CAI than when the cell line is culturedunder the same culture conditions without CAI.

This invention provides for an isolated CAIR protein, wherein theprotein specifically binds to an antibody generated against an immunogenconsisting of the amino acid sequence depicted by SEQ ID NO:2. Morespecifically the carboxyl terminus of the protein consists of apolypeptide of SEQ ID NO:2. In addition, the protein may berecombinantly produced. This protein is expressed at higher levels in acell line cultured in the presence of at least 10 μM concentration ofCAI than in the cell line cultured under the same culture conditionswithout CAI.

In still another embodiment, this invention provides for an isolatednucleic acid encoding a CAIR resistance protein, wherein the proteinspecifically binds to an antibody generated against an immunogenconsisting of the amino acid sequence depicted by SEQ ID NO:2. Morespecifically this nucleic acid comprises the nucleotide sequence of SEQID NO:1.

In yet another embodiment, this invention provides for cells capable ofgrowing and proliferating when cultured in the presence of CAI rangingin concentration from 1 μM to 45 μM, more particularly in CAI ranging inconcentration from 20 μM to 45 μM, and still more particularly in CAIranging in concentration from 40 μM to 45 μM. Even more particularlythese cells are A2058-20R cells or OVCAR3-R cells.

This invention additionally provides for a method of determiningresistance of CAI of a biological sample where the method comprises thesteps of a) contacting a binding agent capable of specifically binding aCAI resistance protein to the biological sample; b) incubating thebinding agent with the biological sample to form a binding agent:CAIRprotein complex; and c) detecting the complex. More particularly, thebinding agent is an antibody that is specifically immunoreactive withthe CAI resistance protein. Even more particularly, the step ofdetecting comprises a) contacting the complex with a labeled antibodythat specifically binds the binding agent; and b) detecting the labeledantibody.

In another embodiment, this invention provides for a method ofdetermining resistance of CAI of a biological sample where the methodcomprises the steps of a) contacting a binding agent capable ofspecifically binding nucleotide sequence that encodes a CAI resistanceprotein with the biological sample; b) incubating the binding agent withthe biological sample to form a binding agent:nucleic acid complex; andc) detecting the complex. More particularly, the binding agent is anucleic acid that hybridizes specifically to a second nucleic acidsequence that encodes a CAI resistance protein under stringentconditions. Even more particularly, the nucleic acid hybridizesspecifically to a DNA sequence of SEQ ID NO:1. Still more particularly,the step of detecting comprises detecting a labeled nucleic acid.

This invention provides for a kit for determining the resistance to CAIwhere the kit comprises a container containing a binding agent capableof specifically binding a CAIR protein. More particularly, the bindingagent of the kit is an antibody that specifically binds to a CAIRprotein. Even more particularly, the kit further comprises a containercontaining a means for detecting the antibody.

In another embodiment, the invention provides for a kit for determiningresistance to CAI of a biological sample where the kit comprises acontainer containing a binding agent capable of specifically binding anucleic acid sequence encoding a CAI resistance protein. Moreparticularly, the binding agent is a second nucleic acid that hybridizesspecifically to the nucleic acid sequence under stringent conditions.

The invention also provides for a kit for assaying compounds for a CAIresistance obviating activity, where the kit comprises a containercontaining a CAI resistant cell. More particularly the cell is A2058-20Ror OVCAR3-R.

The invention provides for an antibody that specifically binds to a CAIRprotein. More particularly, these antibody is generated against animmunogen consisting of the amino acid sequence depicted by SEQ ID NO:2.

In another embodiment, this invention provides for a pharmacologicalcomposition that reduces CAI resistance said composition comprising abinding agent that binds to a nucleotide encoding a CAIR protein. Moreparticularly the binding agent is an antisense molecule that hybridizesspecifically to a nucleotide encoding a CAIR protein. Even moreparticularly the nucleotide encoding a CAIR protein is SEQ ID NO:1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Northern analyses indicating up-regulation of the CAIRgene. The upper left gel shows a Northern blot of total RNA fromparental A2058 cells hybridized to a probe obtained from a subtractivehybridization of A2058-20R CAI resistant cells against parental A2058cells. A cDNA library was created from A2058-20R cells and screened withthe subtracted probe. The selected clones when hybridized againstNorthern blots identified altered gene expression in response to chronicexposure to CAI. The gels labeled 21 DBB (CAIR-1), 15 CBB (CAIR-3) and13 BAA (CAIR-2) represent clones showing increased expression inresponse to CAI exposure. The transcribed message of CAIR-1 is about 2.8kb, while the messages for CAIR3 and CAIR-2 are about 4.5 kb and 4.2 kbrespectively. In all cases, the blots were stripped and thenrehybridized with radiolabeled glyceraldehyde-3-phosphate dehydrogenaseGAPDH as a housekeeping gene to correct for differential loading (lowergels).

DESCRIPTION OF THE PREFERRED EMBODIMENT Definitions

Abbreviations for the twenty naturally occurring amino acids followconventional usage. In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

The term "CAI resistance" as used herein refers to the ability of a cellto grow and proliferate in the presence of carboxyamido-triazole (CAI)and functionally equivalent compositions at a concentration of 10 μM orgreater. Functionally equivalent compositions are those compositionsthat act in a manner identical to or analogous to CAI in inhibitingcalcium influx. Although the specific activity levels of thefunctionally equivalent compositions may differ, they produce the samebiological effects. These include inhibition of calcium influx andconsequent reduction in cell proliferation rate. Examples offunctionally equivalent compositions include the antimycotic imidizolessuch as ketoconazole, miconazole, fluconazole, econazole anditraconazole, the compound described by Merritt, et al., Biochem. J.,271: 515-522 (1990) and its analogs. These analogs, described incopending patent application U.S. Ser. No. 07/985,402, now issued asU.S. Pat. No. 5,359,078, include compounds of the formula: ##STR1##where the group Ar¹ is typically a hydrophobic radical including, butnot limited to phenyl, trioxaadamantyl, anthracenyl, anthraquinonyl,naphthyl, and phenanthryl. The radicals in this group may also be halosubstituted. Additionally, Ar¹ may be --Ar² --X--Ar³ where Ar² and Ar³are typically aromatic groups including phenyl, substituted phenyl,naphthyl, and halo substituted naphthyl. Ar² and Ar³ may be the same ordifferent. The symbol X represents a linking group and may be O, S, SO₂,CO, CHCN, straight chain alkyl, alkoxy, and alkoxyalkyl. The group Zrepresents a nitrogen-containing heterocycle. Example ofnitrogen-containing heterocycles for this group are imidazolyl,1,2,3-triazolyl, 1,2,4-triazolyl, pyrazinyl, purinyl, pyrimidinyl,1,2,3-triazolo-{4,5-d}-pyrimidinyl, and their halo-substituted analogs.The symbol p represents an integer of from 0 to 4.

A "CAI resistance protein" or "CAIR protein" refers to a protein whoseexpression in a cell is correlated with the ability of a cell to displayCAI resistance. That is, to grow and proliferate in the presence of CAIand functionally equivalent compositions. The CAIR-1 resistance proteinof this invention is characterized by at least one proline rich domain,a novel domain homologous to the Src homology 3 (SH3) binding domainconsensus sequence. The SH3 binding protein domain consensus sequence isXPXXPPPψXP SEQ ID NO:3 where positions 2, 7 and 10 are obligate P(proline), the X is any amino acid, and the ψ is a hydrophobic aminoacid. The CAIR proteins of this invention are up-regulated when the cellis exposed to CAI and this up-regulation is characteristic of andcorrelated with CAI resistance.

The term "nucleic acid" refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, would encompass known analogs of naturalnucleotides that can function in a similar manner as naturally occurringnucleotides.

The phrase "nucleic acid encoding" or "nucleic acid sequence encoding"refers to a nucleic acid which directs the expression of a specificprotein or peptide. The nucleic acid sequences include both the DNAstrand sequence that is transcribed into RNA and the RNA sequence thatis translated into protein. The nucleic acid sequences include both fulllength nucleic acid sequences as well as shorter sequences derived fromthe full length sequences. It is understood that a particular nucleicacid sequence includes the degenerate codons of the native sequence orsequences which may be introduced to provide codon preference in aspecific host cell. The nucleic acid includes both the sense andantisense strands as either individual single strands or in the duplex.The terms "hybridize" or "hybridizing" refer to the binding of twosingle stranded nucleic acids via complementary base pairing.

The phrase "hybridizing specifically to", refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in apreparation of total cellular DNA or RNA.

The term "stringent conditions" refers to conditions under which a probewill hybridize to its target subsequence, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. Generally, stringent conditions are selected to beabout 5° C. lower than the thermal melting point (Tm) for the specificsequence at a defined ionic strength and pH. The Tm is the temperature(under defined ionic strength and pH) at which 50% of the targetsequence hybridizes to a complementary probe. Typically, stringentconditions will be those in which the salt concentration is at leastabout 0.1 to 1.0 N Na ion concentration at pH 7.0 to 7.5 and thetemperature is at least about 60° C. for long sequences (e.g. greaterthan about 50 nucleotides) and at least about 42° C. for shortersequences (e.g. 10 to 50 nucleotides).

The terms "isolated" or "substantially pure" when referring to nucleicacid sequences encoding CAIR proteins or fragments thereof refers toisolated nucleic acids which do not encode proteins or peptides otherthan CAIR proteins or peptides.

The terms "isolated" or "substantially purified" or "isolated" whenreferring to a CAIR proteins, means a chemical composition which isessentially free of other cellular components. It is preferably in ahomogeneous state although it can be in either a dry or aqueoussolution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. Generally, a substantially purified or isolatedprotein will comprise more than 80% of all macromolecular speciespresent in the preparation. Preferably, the protein is purified torepresent greater than 90% of all macromolecular species present. Morepreferably the protein is purified to greater than 95%, and mostpreferably the protein is purified to essential homogeneity, whereinother macromolecular species are not detected by conventionaltechniques.

The phrase "specifically binds to an antibody" or "specificallyimmunoreactive with", when referring to a protein or peptide, refers toa binding reaction which is determinative of the presence of the proteinin the presence of a heterogeneous population of proteins and otherbiologics. Thus, under designated immunoassay conditions, the specifiedantibodies bind to a particular protein and do not bind in a significantamount to other proteins present in the sample. Specific binding to anantibody under such conditions may require an antibody that is selectedfor its specificity for a particular protein. For example, antibodiesraised to the CAI protein fragment with the amino acid sequence depictedin SEQ ID NO:2 can be selected to obtain antibodies specificallyimmunoreactive with CAIR proteins and not with other proteins. Theseantibodies recognize proteins homologous to the CAIR protein. Homologousproteins encompass the family of CAIR proteins, but do not include othersignal transduction proteins that are not up-regulated in response toexposure to CAI. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein. See Harlow andLane (1988) Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York, for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.

The term "binding agent:CAIR protein complex", as used herein, refers toa complex of a binding agent and a CAIR protein that is formed byspecific binding of the binding agent to the CAIR protein. Specificbinding of the binding agent means that the binding agent has a specificbinding site that recognizes a site on the CAIR protein. For example,antibodies raised to a CAIR protein and recognizing an epitope on theCAIR protein are capable of forming a binding agent:CAIR protein complexby specific binding. Typically, the formation of a binding agent:CAIRprotein allows the measurement of CAIR protein in a mixture of otherproteins and biologics. The term "antibody:CAIR protein complex" refersto a binding agent:CAIR protein complex in which the binding agent is anantibody.

The term "binding agent:nucleic acid complex", as used herein, refers toa complex of a binding agent and a nucleic acid that is formed byspecific binding of the binding agent to the nucleic acid. Specificbinding of the binding agent means that the binding agent has a specificbinding site that recognizes a site on the CAIR protein. For example,nucleic acid probes complementary to a region of the nucleic acidsequence encoding a CAIR protein are capable for forming a probe:CAInucleic acid complex.

The term "biological sample" as used herein refers to any sampleobtained from a living organism or from an organism that has died.Examples of biological samples include body fluids, tissue specimens,and tissue culture lines taken from patients.

The term "recombinant DNA" or "recombinantly produced DNA" refers to DNAwhich has been isolated from its native or endogenous source andmodified either chemically or enzymatically to deletenaturally-occurring flanking nucleotides or provide flanking nucleotidesthat do not naturally occur. Flanking nucleotides are those nucleotideswhich are either upstream or downstream from the described sequence orsub-sequence of nucleotides.

The term "recombinant protein" or "recombinantly produced protein"refers to a peptide or protein produced using non-native cells that donot have an endogenous copy of DNA able to express the protein. Thecells produce the protein because they have been genetically altered bythe introduction of the appropriate nucleic acid sequence. Therecombinant protein will not be found in association with proteins andother subcellular components normally associated with the cellsproducing the protein.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: "referencesequence", "comparison window", "sequence identity", and "percentage ofsequence identity".

A "reference sequence" is a defined sequence used as a basis for asequence comparison; a reference sequence may be a subset of a largersequence, for example, as a segment of a full-length cDNA or genesequence given in a sequence listing, such as the nucleic acid sequenceof SEQ ID NO:1, or may comprise a complete cDNA or gene sequence.

"Percentage of sequence identity" is determined by comparing twooptimally aligned sequences or subsequences over a comparison window orspan, wherein the portion of the polynucleotide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalsubunit (e.g. nucleic acid base or amino acid residue) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. Percentage sequence identity when calculated usingthe programs GAP or BESTFIT (see below) is calculated using default gapweights.

When percentage of sequence identity is used in reference to proteins orpeptides it is recognized that residue positions which are not identicalmay differ by conservative amino acid substitutions, where amino acidsresidues are substituted for other amino acid residues with similarchemical properties (e.g. charge or hydrophobicity) and therefore do notchange the functional properties of the molecule. Where sequences differin conservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch thereby increasingthe percentage sequence identity. Thus, for example, where an identicalamino acid is given a score of 1 and a non-conservative substitution isgiven a score of zero, a conservative substitution is given a scorebetween zero and 1. The scoring of conservative substitutions iscalculated according to the algorithm of Meyers and Miller, ComputerApplic. Biol. Sci., 4: 11-17 (1988) as implemented in the programPC/GENE (Intelligenetics, Moutain View, Calif., U.S.A.). The followingsix groups each contain amino acids that are conservative substitutionsfor one another:

1) Alanine (A), Serine (S), Threonine (I);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

A "comparison window", as used herein, refers to a segment of at leastabout 20 contiguous positions, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Methods of alignment of sequences for comparison are well known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981) which is incorporated herein by reference, by the homologyalignment algorithm of Needleman and Wunsch J. Mol. Biol. 48: 443 (1970)which is incorporated herein by reference, by the search for similaritymethod of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444(1988) which is incorporated herein by reference, by computerizedimplementations of these algorithms (including, but not limited toCLUSTAL in the PC/Gene program by Intelligenetics, Mountain View,Calif., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group (GCG), 575 Science Dr.,Madison, Wis., U.S.A.), or by inspection. In particular, methods foraligning sequences using the CLUSTAL program are well described byHiggins and Sharp in Gene, 73: 237-244 (1988) and in CABIOS 5: 151-153(1989) both of which are incorporated herein by reference.

DETAILED DESCRIPTION

This invention provides for isolated proteins correlated with cellularresistance to carboxyamido-triazole (CAI) and functionally equivalentcompositions, and for isolated nucleic acids that encode these proteins.These isolated nucleic acid and protein compositions can be used in anumber of applications. For instance, both the protein and the nucleicacid can be used as markers indicating the acquisition of resistance toCAI by tumor cells undergoing CAI chemotherapy. The nucleic acidsequence may also be used as a selectable marker by linking it toanother nucleic acid sequence in a vector and transforming a cell line.The transformed cells may be selected by exposing the cells to CAI andselecting those cells that survive.

In addition, at least one CAIR protein, CAIR-1, contains Src homology 3(SH3) binding domains which interact with SH3 domains on a variety ofproteins and may be used to modulate or inactivate various intracellularsignaling pathways or cytoskeletal elements. Thus, the protein orfragments bearing SH3 binding domains may be used as therapeutics fordisease states characterized by calcium signaling problems. Furthermore,the protein or fragments bearing SH3 binding domains may be used toassay for and identify other proteins carrying SH3 domains and therebyidentify new elements in the signalling pathway.

The present invention also provides for antibodies specific to the CAIRproteins. These antibodies may be used to inactivate the protein andthereby obviate CAI resistance. In addition, antibodies that bind theSH3 binding domains may be used to compete with SH3 bearing proteins andthus used to modulate or inhibit various signaling pathways or theaction of various cytoskeletal elements.

This invention additionally provides for cell lines that display CAIresistance. These cell lines may be used as an assay to screen forcompounds that can reduce or eliminate CAI resistance. They can also beused as model systems for evaluating the effectiveness of mixed drugregimens that include CAI or analogous calcium regulation inhibitors.

A. Inducing Expression of CAIR Genes

CAIR genes are up-regulated in cells that grow and proliferate when theyare challenged with CAI and functionally equivalent compositions. Thesecompounds include the antimycotic imidizoles such as ketoconazole,miconazole, fluconazole, econazole and itraconazole, the compounddescribed by Merritt, et al., Biochem. J., 271: 515-522 (1990) and itsanalogs as described above.

CAIR genes may be identified by culturing cells in the presence ofincreasing concentrations of CAI and its functional equivalents. Cellsthat grow and proliferate are selected, their RNA isolated and asubtraction hybridization is performed against the unchallenged parentalcell line. Subtractive hybridization yields a probe which may be labeledand is then used to probe cDNA libraries for clones showing elevatedexpression of the gene in the presence of CAI or its functionalequivalents.

B. CAIR Proteins

The CAI resistance (CAIR) proteins are correlated with cellular CAIresistance. These expression of these proteins is typically up-regulatedin cells that survive a challenge with CAI or functionally equivalentcompounds.

The carboxyl terminus of one such protein (CAIR-1) is shown in SEQ IDNO:2. This protein contains a number of Src homology 3 domain (SH3)binding protein domains. SH3 domains are conserved among a very largegroup of proteins including cytoskeletal elements and intracellularsignaling proteins (Ren, et al., Science 259: 1157-1161 (1993)). Thesedomains appear to be involved in the mediation of protein-proteinassociations and regulate cytoplasmic signaling. Id.

Thus the CAIR-1 protein of this invention or fragments thereofcomprising the SH3 binding proteins domains may be used in inhibit ormodulate various intracellular signaling pathways. This provides a modeof treatment for various diseases characterized by abnormalintracellular signaling such as cancer, hyperproliferative states,correction of collagen vascularities (e.g. lupis and rheumatoidarthritis), nephropathies, neuropathies (e.g. ALF, Alzheimers disease),and myopathies (e.g. cardiomyopathies, skeletalmyopathies).

Means of identifying SH3 binding domains in CAIR proteins are well knownto those of skill in the art. Where the protein sequence is known,regions of the protein whose sequences fulfill the consensus definitionfor SH3 binding domains (XPXXPPPψXP (SEQ ID NO:3) where positions 2, 7,and 10 are obligate P (proline), the X is any amino acid, and the ψ is ahydrophobic amino acid) may easily be identified either by inspection ofthrough the use of sequence analysis programs such as FASTA or BESTFIT.

SH3 binding sites may also be identified by assaying for actual bindingbetween the CAIR protein and proteins bearing known SH3 sites such asAbl, Fyn, Lck and the p85 α subunit of phosphatidylinositol 3' kinase.Protein binding assays are well known to those of skill in the art. In apreferred embodiment, the assay may be conducted by creating aGST-CAIR-1 fusion protein, immobilizing the protein onglutathione-Sepharose beads, exposing the immobilized protein to thescreening proteins (e.g., Abl, Fyn, Lck and p85) and then assaying forbinding of the screening proteins. Binding may easily be assayed usinglabeled monoclonal antibodies specific to the SH3 proteins. These arecommercially available. An example of this assay is provided in Example5.

The CAIR proteins of the present invention also include proteins thatmay contain various amino acid substitutions, but retain essentially thesame conformation and activity as the unmodified protein. Thus, CAIR-1protein, the carboxyl terminal sequence of which is listed in SEQ IDNO:2, also embraces proteins reflecting conservative amino acidsubstitutions in the listed sequence. These related proteins may bedetermined by their sequence identity with the CAIR-1 protein of SEQ IDNO:2. The CAIR proteins of this invention have at least 80 percentsequence identity, preferably at least 90 percent sequence identity, andmore preferably at least 95 percent sequence identity as compared to areference CAIR protein (e.g. SEQ ID NO:2).

Alternatively, related CAIR proteins may be determined by theircrossreactivity with antibodies generated against a defined immunogenencoded by the CAIR-1 protein of the present invention. A CAIR proteinthat specifically binds to or that is specifically immunoreactive to anantibody generated against a defined immunogen, such as an immunogenconsisting of the amino acid sequence of SEQ ID NO:2, is determined inan immunoassay. The immunoassay uses a polyclonal antiserum which wasraised to the protein of SEQ ID NO:2. This antiserum is selected to havelow crossreactivity against other non CAIR proteins and any suchcrossreactivity is removed by immunoabsorbtion prior to use in theimmunoassay.

In order to produce antisera for use in an immunoassay, the protein ofSEQ ID NO:2 is isolated as described herein (e.g. the recombinantprotein is produced in a mammalian cell line). Then, a mammal (e.g. aninbred strain of mouse such as balb/c) is immunized with the protein ofSEQ ID NO:2 using a standard adjuvant, such as Freund's adjuvant, and astandard immunization protocol (see Harlow and Lane, supra).Alternatively, a synthetic peptide derived from the sequences disclosedherein and conjugated to a carrier protein can be used an immunogen. Forinstance, the peptide of SEQ ID NO:2 may be used. Polyclonal sera arecollected and titered against the immunogen protein in an immunoassay,for example, a solid phase immunoassay with the immunogen immobilized ona solid support. Polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-CAIRproteins, using a competitive binding immunoassay such as the onedescribed in Harlow and Lane, supra, at pages 570-573.

Immunoassays in the competitive binding format can be used for thecrossreactivity determinations. For example, the protein of SEQ ID NO:2can be immobilized to a solid support. Proteins added to the assaycompete with the binding of the antisera to the immobilized antigen. Theability of the above proteins to compete with the binding of theantisera to the immobilized protein is compared to the protein of SEQ IDNO:2. The percent crossreactivity for the above proteins is calculated,using standard calculations. Those antisera with less than 10%crossreactivity with each of the proteins listed above are selected andpooled. The cross-reacting antibodies are then removed from the pooledantisera by immunoabsorbtion with the above-listed proteins.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein tothe immunogen protein (the CAIR protein of SEQ ID NO:2). In order tomake this comparison, the two proteins are each assayed at a wide rangeof concentrations and the amount of each protein required to inhibit 50%of the binding of the antisera to the immobilized protein is determined.If the amount of the second protein required is less than 10 times theamount of the protein of SEQ ID NO:2 that is required, then the secondprotein is said to specifically bind to an antibody generated to animmunogen consisting of the protein of SEQ ID NO:2.

It is understood that CAIR proteins refer to a family of relatedproteins. For a particular gene product, such as CAIR-1 protein, theterm refers not only to the amino acid sequences disclosed herein, butalso to other proteins that are allelic, non-allelic or speciesvariants. It also understood that the term "CAIR proteins" includesnonnatural mutations introduced by deliberate mutation usingconventional recombinant technology such as single site mutation or byexcising short sections of DNA encoding CAIR proteins or by substitutingnew amino acids or adding new amino acids. Such minor alterations mustsubstantially maintain the immunoidentity of the original moleculeand/or its biological activity. Thus, these alterations include proteinsthat are specifically immunoreactive with a designated naturallyoccurring CAIR protein, for example, the CAIR-1 protein shown in SEQ IDNO:2. The biological properties of the altered proteins can bedetermined by expressing the protein in an appropriate cell line andchallenging the cell in culture with CAI. Particular proteinmodifications considered minor would include conservative substitutions,that is substitution of amino acids of similar chemical properties,e.g., glutamic acid for aspartic acid or glutamine for asparagine. Byaligning a protein optimally with the protein of SEQ ID NO:2 and byusing the conventional immunoassays described herein to determineimmunoidentity cell growth assays to determine biological activity, onecan readily determine the protein compositions of the invention.

CAIR proteins designated by their tissue of origin refer to thegene-product from this family that is predominantly expressed in thattissue. For instance, the term "skeletal muscle CAIR protein" refers tothe CAIR protein that is expressed in skeletal tissue. As anotherexample, the term "heart CAIR protein" refers to the CAIR proteinexpressed in heart tissue. Since CAIR proteins represent a family ofrelated proteins, the proteins expressed in different tissues can be theproduct of different genes in the family.

C. Nucleic Acids Encoding CAIR Proteins

This invention relates to isolated nucleic acid sequences encoding CAIRproteins. The nucleic acid compositions of this invention, whether RNA,cDNA, genomic DNA, or a hybrid of the various combinations, may beisolated from natural sources or may be synthesized in vitro. Thenucleic acids claimed may be present in transformed or transfected wholecells, in a transformed or transfected cell lysate, or in a partiallypurified or substantially pure form.

The nucleic acid sequences of the invention are typically identical toor show at least 85 percent sequence identity, preferably at least 90 to95 percent sequence identity, and more preferably at least 99 percentsequence identity (determined as described above) to the nucleic acidsequence of SEQ ID NO:1. Nucleic acids encoding mammalian CAIR-1proteins will typically hybridize to the nucleic acid sequence of SEQ IDNO:1 under stringent conditions. For example, nucleic acids encodingCAIR proteins will hybridize to the nucleic acid of SEQ ID NO:1 underthe hybridization and wash conditions of 50% formamide at 42° C. Otherstringent hybridization conditions may also be selected. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The Tm is the temperature (under defined ionic strengthand pH) at which 50% of the target sequence hybridizes to a perfectlymatched probe. Typically, stringent conditions will be those in whichthe salt concentration is at least about 0.1 to 1.0 N Na ionconcentration at pH 7.0 to 7.5 and the temperature is at least about 60°C. As other factors may significantly affect the stringency ofhybridization, including, among others, base composition and size of thecomplementary strands, the presence of organic solvents and the extentof base mismatching, the combination of parameters is more importantthan the absolute measure of any one.

Techniques for nucleic acid manipulation of genes encoding the CAIRproteins such as subcloning nucleic acid sequences encoding polypeptidesinto expression vectors, labeling probes, DNA hybridization, and thelike are described generally in Sambrook, et al., Molecular Cloning--ALaboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989, which is incorporated herein byreference. This manual is hereinafter referred to as "Sambrook, et al.".

In general nucleic acids encoding CAIR proteins may be obtained by firstproducing a cell line in which the expression of CAIR proteins areup-regulated. This is accomplished by culturing the cells in thepresence of CAI or a functional equivalent as described in section (A)and in Example 1.

A subtraction probe is then obtained by subtractive hybridizationbetween, CAI resistant cells obtained in the culture and thenon-resistant parental cell line. Methods of subtractive hybridizationare well known to those of skill in the art. See, for example, Hampson,et al., Nucleic Acids Res., 20: 2899 (1992), and Example 2.

The resistant cell line is then used to produce a cDNA library which maythen be probed using the labeled subtraction probe. Positive clones arethen isolated and those clones are screened for up-regulated expressionof CAIR proteins using the labeled subtraction probe. Clones showingup-regulated CAIR protein expression may then be sequenced usingstandard techniques.

SEQ ID NO:1 provides the nucleotide sequence obtained in this manner for1269 bases of the 3' end of the CAIR-1 gene obtained from A2058-20Rcells. This represents about 40% of the full length message which isapproximately 2.8 kb as determined from the Northern blot (FIG. 1). SEQID NO:1 may be used to obtain the full length sequence using numerousmethods well known to those of skill in the art.

For example, various methods of amplifying target sequences, such as thepolymerase chain reaction, can also be used to prepare DNA encoding CAIRresistance protein. Polymerase chain reaction (PCR) technology is usedto amplify such nucleic acid sequences directly from mRNA, from cDNA,and from genomic DNA libraries or cDNA libraries. The isolated sequencesencoding CAIR proteins may also be used as templates for PCRamplification.

In PCR techniques, oligonucleotide primers complementary to either the5' or the 3' borders of the DNA region to be amplified are synthesized,or random primers may be used. The polymerase chain reaction is thencarried out using the two primers. See PCR Protocols: A Guide to Methodsand Applications. (Innis, M, Gelfand, D., Sninsky, J. and White, T.,eds.), Academic Press, San Diego (1990). Primers can be selected toamplify the entire regions encoding a full-length CAIR protein or toamplify smaller DNA segments as desired.

PCR can be used in a variety of protocols to isolate cDNA's encoding theCAIR proteins. Generally appropriate primers and probes for amplifyingDNA encoding CAIR proteins are generated from analysis of the DNAsequences encoding CAIR proteins, such as that found in SEQ ID NO:1. Forexample, the oligonucleotides complementary to regions of SEQ ID NO:1can be used in a PCR protocol to amplify regions of DNA's encoding CAIRproteins. Once such regions are PCR-amplified, they can be sequenced andoligonucleotide probes can be prepared from sequence obtained. Theseprobes can then be used to isolate DNA's encoding CAIR proteins. CAIRproteins can be isolated from a variety of different tissues using thisprocedure.

A preferred approach for DNA isolation is 5' RACE. Briefly, thistechnique involves using PCR to amplify a DNA sequence using a random 5'primer and a defined 3' primer. The amplified sequence is then subclonedinto a vector where it is then sequenced using standard techniques. The5' RACE method is well known to those of skill in the art and kits toperform 5' RACE are commercially available (e.g. 5' RACE System,GibcoBRL, Grand Island, N.Y., U.S.A.).

There are numerous other methods of isolating the DNA sequences encodingthe CAIR resistance proteins. For example, DNA may be isolated from agenomic or cDNA library using labelled oligonucleotide probes havingsequences complementary to the sequences disclosed herein (SEQ ID NO:1).For example, full-length probes may be used, or oligonucleotide probesconsisting of subsequences of SEQ ID NO:1 may be used. Such probes canbe used directly in hybridization assays to isolate DNA encoding CAIRproteins. Alternatively probes can be designed for use in amplificationtechniques such as PCR, and DNA encoding CAIR proteins may be isolatedby using methods such as PCR (see below).

To prepare a cDNA library, mRNA is isolated from tissue such as heart,skeletal muscle, or cell lines that show CAI resistance. cDNA isprepared from the mRNA and ligated into a recombinant vector. The vectoris transfected into a recombinant host for propagation, screening andcloning. Methods for making and screening cDNA libraries are well known.See Gubler and Hoffman, Gene 25:263-269, (1983) and Sambrook, et al.

For a genomic library, the DNA is extracted from the tissue or cell lineand either mechanically sheared or enzymatically digested to yieldfragments of about 12-20 kb. The fragments are then separated bygradient centrifugation from undesired sizes and are constructed inbacteriophage lambda vectors or other vectors. These vectors and phageare packaged in vitro, as described in Sambrook, et al. Recombinantphage are analyzed by plaque hybridization as described in Benton andDavis, Science, 196: 180-182 (1977). Colony hybridization is carried outas generally described in M. Grunstein et al. Proc. Natl. Acad. Sci.U.S.A., 72: 3961-3965 (1975).

DNA encoding an CAIR protein is identified in either cDNA or genomiclibraries by its ability to hybridize with nucleic acid probes, forexample on Southern blots, and these DNA regions are isolated bystandard methods familiar to those of skill in the art. Alternatively,RNA encoding CAIR proteins may be identified by its ability to hybridizewith nucleic acid probes in Northern blots. See Sambrook, et al.

Oligonucleotides for use as probes are chemically synthesized accordingto the solid phase phosphoramidite triester method first described byBeaucage and Carruthers, Tetra. Lett., 22: 1859-1862 (1981) using anautomated synthesizer, as described in Needham-VanDevanter, et al.,Nucleic Acids Res., 12: 6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson and Regnier, J. Chrom.,255: 137-149 (1983). The sequence of the synthetic oligonucleotide canbe verified using the chemical degradation method of Maxam and Gilbert,in Grossman, L. and Moldave, D., eds. Academic Press, New York, Methodsin Enzymology, 65: 499-560 (1980).

Other methods known to those of skill in the art may also be used toisolate DNA encoding the CAIR protein. See Sambrook, et al. for adescription of other techniques for the isolation of DNA encodingspecific protein molecules.

D. Expression of CAIR Proteins

Once DNA encoding CAIR proteins is isolated and cloned, one can expressthe CAIR proteins in a variety of recombinantly engineered cells. It isexpected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of DNA encodingCAIR proteins.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CAIR proteins will typically be achieved by operably linkingthe DNA or cDNA to a promoter (which is either constitutive orinducible), followed by incorporation into an expression vector. Thevectors can be suitable for replication and integration in eitherprokaryotes or eukaryotes. Typical expression vectors containtranscription and translation terminators, initiation sequences, andpromoters useful for regulation of the expression of polynucleotidesequence encoding CAIR proteins. To obtain high level expression of acloned gene, such as those polynucleotide sequences encoding CAIRproteins, it is desirable to construct expression plasmids whichcontain, at the minimum, a strong promoter to direct transcription, aribosome binding site for translational initiation, and atranscription/translation terminator. The expression vectors may alsocomprise generic expression cassettes containing at least oneindependent terminator sequence, sequences permitting replication of theplasmid in both eukaryotes and prokaryotes, i.e., shuttle vectors, andselection markers for both prokaryotic and eukaryotic systems. SeeSambrook et al. Examples of expression of CAIR proteins in bothprokaryotic and eukaryotic systems are described below.

1. Expression in Prokaryotes

A variety of procaryotic expression systems may be used to express CAIRproteins. Examples include E. coli, Bacillus, Streptomyces, and thelike. For example, CAIR proteins may be expressed in E. coli.

It is essential to construct expression plasmids which contain, at theminimum, a strong promoter to direct transcription, a ribosome bindingsite for translational initiation, and a transcription/translationterminator. Examples of regulatory regions suitable for this purpose inE. coli are the promoter and operator region of the E. coli tryptophanbiosynthetic pathway as described by Yanofsky, J. Bacteriol., 158:1018-1024 (1984) and the leftward promoter of phage lambda (Pλ) asdescribed by Herskowitz, et al., Ann. Rev. Genet., 14: 399-445 (1980).The inclusion of selection markers in DNA vectors transformed in E. coliis also useful. Examples of such markers include genes specifyingresistance to ampicillin, tetracycline, or chloramphenicol. See Sambrooket al. for details concerning selection markers for use in E. coli.

CAIR proteins produced by prokaryotic cells may not necessarily foldproperly. During purification from E. coli the expressed protein mayfirst be denatured and then renatured. This can be accomplished bysolubilizing the bacterially produced proteins in a chaotropic agentsuch as guanidine HCl and reducing all the cysteine residues with areducing agent such as beta-mercaptoethanol. The protein is thenrenatured, either by slow dialysis or by gel filtration. See U.S. Pat.No. 4,511,503.

Detection of the expressed antigen is achieved by methods known in theart as radioimmunoassay, or Western blotting techniques orimmunoprecipitation. Purification from E. coli can be achieved followingprocedures described in U.S. Pat. No. 4,511,503.

2. Expression in Eukaryotes

A variety of eukaryotic expression systems such as yeast, insect celllines, bird, fish, and mammalian cells, are known to those of skill inthe art. As explained briefly below, CAIR proteins may be expressed inthese eukaryotic systems.

Synthesis of heterologous proteins in yeast is well known. Methods inYeast Genetics, Sherman, et al., Cold Spring Harbor Laboratory, (1982)is a well recognized work describing the various methods available toproduce the protein in yeast.

Suitable vectors usually have expression control sequences, such aspromoters, including 3-phosphoglycerate kinase or other glycolyticenzymes, and an origin of replication, termination sequences and thelike as desired. For instance, suitable vectors are described in theliterature (Botstein, et al., 1979, Gene, 8:17-24; Broach, et al., 1979,Gene, 8:121-133).

Two procedures are used in transforming yeast cells. In one case, yeastcells are first converted into protoplasts using zymolyase, lyticase orglusulase, followed by addition of DNA and polyethylene glycol (PEG).The PEG-treated protoplasts are then regenerated in a 3% agar mediumunder selective conditions. Details of this procedure are given in thepapers by J. D. Beggs, 1978, Nature (London), 275: 104-109; and Hinnen,et al., Proc. Natl. Acad. Sci. U.S.A., 75: 1929-1933 (1978). The secondprocedure does not involve removal of the cell wall. Instead the cellsare treated with lithium chloride or acetate and PEG and put onselective plates (Ito, et al., J. Bact., 153: 163-168 (1983)).

CAIR proteins, once expressed, can be isolated from yeast by lysing thecells and applying standard protein isolation techniques to the lysates.The monitoring of the purification process can be accomplished by usingWestern blot techniques or radioimmunoassay of other standardimmunoassay techniques.

The sequences encoding CAIR proteins can also be ligated to variousexpression vectors for use in transforming cell cultures of, forinstance, mammalian, insect, bird or fish origin. Illustrative of cellcultures useful for the production of the polypeptides are mammaliancells. Mammalian cell systems often will be in the form of monolayers ofcells although mammalian cell suspensions may also be used. A number ofsuitable host cell lines capable of expressing intact proteins have beendeveloped in the art, and include the HEK293, BHK21, and CHO cell lines,and various human cells such as COS cell lines, HeLa cells, myeloma celllines, Jurkat cells, etc. Expression vectors for these cells can includeexpression control sequences, such as an origin of replication, apromoter (e.g., the CMV promoter, a HSV tk promoter or pgk(phosphoglycerate kinase) promoter), an enhancer (Queen et al., Immunol.Rev. 89: 49 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site), and transcriptional terminatorsequences. Other animal cells useful for production of CAIR proteins areavailable, for instance, from the American Type Culture CollectionCatalogue of Cell Lines and Hybridomas (7th edition, 1992).

Appropriate vectors for expressing CAIR proteins in insect cells areusually derived from the SF9 baculovirus. Suitable insect cell linesinclude mosquito larvae, silkworm, armyworm, moth and Drosophila celllines such as a Schneider cell line (See Schneider J. Embryol. Exp.Morphol. 27: 353-365 (1987).

As indicated above, the vector, e.g., a plasmid, which is used totransform the host cell, preferably contains DNA sequences to initiatetranscription and sequences to control the translation of the protein.These sequences are referred to as expression control sequences.

As with yeast, when higher animal host cells are employed,polyadenylation or transcription terminator sequences from knownmammalian genes need to be incorporated into the vector. An example of aterminator sequence is the polyadenylation sequence from the bovinegrowth hormone gene. Sequences for accurate splicing of the transcriptmay also be included. An example of a splicing sequence is the VP1intron from SV40 (Sprague, et al., J. Virol. 45: 773-781 (1983)).

Additionally, gene sequences to control replication in the host cell maybe incorporated into the vector such as those found in bovine papillomavirus type-vectors. Saveria-Campo, Bovine Papilloma virus DNA aEukaryotic Cloning Vector", pages 213-238 in DNA Cloning Vol. II aPractical Approach Ed. D. M. Glover, IRL Press, Arlington, Va. (1985).

The host cells are competent or rendered competent for transformation byvarious means. There are several well-known methods of introducing DNAinto animal cells. These include: calcium phosphate precipitation,fusion of the recipient cells with bacterial protoplasts containing theDNA, treatment of the recipient cells with liposomes containing the DNA,DEAE dextran, electroporation and micro-injection of the DNA directlyinto the cells.

The transformed cells are cultured by means well known in the art.Biochemical Methods in Cell Culture and Virology, Kuchler, R. J.,Hutchinson and Ross, Inc., (1977). The expressed polypeptides areisolated from cells grown as suspensions or as monolayers. The latterare recovered by well known mechanical, chemical or enzymatic means.

E. Purification of CAIR Proteins

The polypeptides produced by recombinant DNA technology may be purifiedby standard techniques well known to those of skill in the art.Recombinantly produced polypeptides can be directly expressed orexpressed as a fusion protein. The protein is then purified by acombination of cell lysis (e.g., sonication) and affinitychromatography. For fusion products, subsequent digestion of the fusionprotein with an appropriate proteolytic enzyme releases the desiredpolypeptide.

The polypeptides of this invention may be purified to substantial purityby standard techniques well known in the art, including selectiveprecipitation with such substances as ammonium sulfate, columnchromatography, immunopurification methods, and others. See, forinstance, R. Scopes, Protein Purification: Principles and Practice,Springer-Verlag: New York (1982), incorporated herein by reference. Forexample, antibodies may be raised to the CAIR proteins as describedherein. Cell membranes are isolated from a cell line expressing therecombinant protein, the protein is extracted from the membranes andimmunoprecipitated. The proteins may then be further purified bystandard protein chemistry techniques as described above.

F. Detection of Nucleotides Encoding CAIR Proteins

The present invention provides methods for detecting DNA or RNA encodingCAIR proteins and for measuring the proteins by immunoassay techniques.These methods are useful for two general purposes. First, assays fordetection of nucleic acids encoding CAIR proteins are useful for theisolation of these nucleic acids from a variety of vertebrate speciesaccording to the methods described in section (C) above and by use ofthe nucleic acid hybridization assays described below. Second, assaysfor the detection of nucleic acids encoding CAIR proteins provide ameans for detecting CAI resistance in a biological sample.

A variety of methods for specific DNA and RNA measurement using nucleicacid hybridization techniques are known to those of skill in the art.See Sambrook, et al. For example, one method for evaluating the presenceor absence of DNA encoding CAIR proteins in a sample involves a Southerntransfer.

Briefly, the digested genomic DNA is run on agarose slab gels in bufferand transferred to membranes. Hybridization is carried out using thenucleic acid probes discussed above. As described above, nucleic acidprobes are designed based on the nucleic acid sequences encoding CAIRproteins (See SEQ ID NO:1). The probes can be full length or less thanthe full length of the nucleic acid sequence encoding the CAIR protein.Shorter probes are empirically tested for specificity. Preferablynucleic acid probes are 20 bases or longer in length. (See Sambrook, etal. for methods of selecting nucleic acid probe sequences for use innucleic acid hybridization.) Visualization of the hybridized portionsallows the qualitative determination of the presence or absence of DNAencoding CAIR proteins.

Similarly, a Northern transfer may be used for the detection of mRNAencoding CAIR proteins. In brief, the mRNA is isolated from a given cellsample using, for example, an acid guanidinium-phenol-chloroformextraction method. The mRNA is then electrophoresed to separate the mRNAspecies and the mRNA is transferred from the gel to a nitrocellulosemembrane. As with the Southern blots, labeled probes are used toidentify the presence or absence of CAIR proteins.

A variety of nucleic acid hybridization formats are known to thoseskilled in the art. For example, common formats include sandwich assaysand competition or displacement assays. Hybridization techniques aregenerally described in "Nucleic Acid Hybridization, A PracticalApproach," Ed. Hames, B. D. and Higgins, S. J., IRL Press, (1985); Galland Pardue Proc. Natl. Acad. Sci., U.S.A., 63: 378-383 (1969); and Johnet al., Nature, 223: 582-587 (1969).

For example, sandwich assays are commercially useful hybridizationassays for detecting or isolating nucleic acid sequences. Such assaysutilize a "capture" nucleic acid covalently immobilized to a solidsupport and a labelled "signal" nucleic acid in solution. The clinicalsample will provide the target nucleic acid. The "capture" nucleic acidand "signal" nucleic acid probe hybridize with the target nucleic acidto form a "sandwich" hybridization complex. To be effective, the signalnucleic acid cannot hybridize with the capture nucleic acid.

Typically, labelled signal nucleic acids are used to detecthybridization. Complementary nucleic acids or signal nucleic acids maybe labelled by any one of several methods typically used to detect thepresence of hybridized polynucleotides. The most common method ofdetection is the use of autoradiography with ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C, or³² P-labelled probes or the like. Other labels include ligands whichbind to labelled antibodies, fluorophores, chemiluminescent agents,enzymes, and antibodies which can serve as specific binding pair membersfor a labelled ligand.

Detection of a hybridization complex may require the binding of a signalgenerating complex to a duplex of target and probe polynucleotides ornucleic acids. Typically, such binding occurs through ligand andanti-ligand interactions as between a ligand-conjugated probe and ananti-ligand conjugated with a signal.

The label may also allow indirect detection of the hybridizationcomplex. For example, where the label is a hapten or antigen, the samplecan be detected by using antibodies. In these systems, a signal isgenerated by attaching fluorescent or enzyme molecules to the antibodiesor, in some cases, by attachment to a radioactive label. (Tijssen, P.,"Practice and Theory of Enzyme Immunoassays," Laboratory Techniques inBiochemistry and Molecular Biology, Burdon, R. H., van Knippenberg, P.H., Eds., Elsevier (1985), pp. 9-20.)

The sensitivity of the hybridization assays may be enhanced through useof a nucleic acid amplification system which multiplies the targetnucleic acid being detected. Examples of such systems include thepolymerase chain reaction (PCR) system and the ligase chain reaction(LCR) system. Other methods recently described in the art are thenucleic acid sequence based amplification (NASBA™, Cangene, Mississauga,Ontario) and Q Beta Replicase systems.

An alternative means for determining the level of expression of a geneencoding an CAIR protein is in situ hybridization. In situ hybridizationassays are well known and are generally described in Angerer, et al.,Methods Enzymol., 152: 649-660 (1987). In an in situ hybridizationassay, cells or tissue specimens are fixed to a solid support, typicallya glass slide. If DNA is to be probed, the cells are denatured with heator alkali. The cells are then contacted with a hybridization solution ata moderate temperature to permit annealing of labeled probes specific toCAIR proteins. The probes are preferably labelled with radioisotopes orfluorescent reporters.

G. Detection of CAIR Proteins by Immunoassay

In addition to detecting expression of nucleic acids encoding CAIRproteins by nucleic acid hybridization, one can also use immunoassays todetect the proteins. Immunoassays can be used to qualitatively orquantitatively analyze for the proteins. A general overview of theapplicable technology can be found in Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Pubs., New York (1988),incorporated herein by reference.

1. Antibody Production

A number of immunogens may be used to produce antibodies specificallyreactive with CAIR proteins. Recombinant protein is the preferredimmunogen for the production of monoclonal or polyclonal antibodies.Naturally occurring protein may also be used either in pure or impureform. Synthetic peptides made using CAIR-1 sequences described hereinmay also used as an immunogen for the production of antibodies to theprotein. Recombinant protein can be expressed in eukaryotic orprokaryotic cells as described above, and purified as generallydescribed above. The product is then injected into an animal capable ofproducing antibodies. Either monoclonal or polyclonal antibodies may begenerated, for subsequent use in immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. In brief, an immunogen, preferably a purified protein,is mixed with an adjuvant and animals are immunized. The animal's immuneresponse to the immunogen preparation is monitored by taking test bleedsand determining the titer of reactivity to the CAIR protein. Whenappropriately high titers of antibody to the immunogen are obtained,blood is collected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to theprotein can be done if desired. (See Harlow and Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (See, Kohler and Milstein, Eur. J. Immunol. 6: 511-519(1976), incorporated herein by reference). Alternative methods ofimmortalization include transformation with Epstein Barr Virus,oncogenes, or retroviruses, or other methods well known in the art.Colonies arising from single immortalized cells are screened forproduction of antibodies of the desired specificity and affinity for theantigen, and yield of the monoclonal antibodies produced by such cellsmay be enhanced by various techniques, including injection into theperitoneal cavity of a vertebrate host. Alternatively, one may isolateDNA sequences which encode a monoclonal antibody or a binding fragmentthereof by screening a DNA library from human B cells according to thegeneral protocol outlined by Huse, et al. Science 246: 1275-1281 (1989).

Methods of production of synthetic peptides are known to those of skillin the art. Briefly, the predicted immunogenic regions of the CAIRprotein sequences described herein are identified. Peptides preferablyat least 10 amino acids in length are synthesized corresponding to theseregions and the peptides are conjugated to larger protein molecules forsubsequent immunization. Preferably, peptide sequences corresponding tounique regions of an CAIR proteins are used to generate antibodiesspecifically immunoreactive with the CAIR proteins. Production ofmonoclonal or polyclonal antibodies is then carried out as describedabove.

2. Immunoassays

A particular protein can be measured by a variety of immunoassaymethods. For a review of immunological and immunoassay procedures ingeneral, see Basic and Clinical Immunology 7th Edition, D. Stites and A.Terr ed. (1991). Moreover, the immunoassays of the present invention canbe performed in any of several configurations, which are reviewedextensively in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, BocaRaton, Fla. (1980); "Practice and Theory of Enzyme Immunoassays," P.Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology,Elsevier Science Publishers B.V. Amsterdam (1985); and, Harlow and Lane,Antibodies, A Laboratory Manual, supra, each of which is incorporatedherein by reference.

Immunoassays for measurement of CAIR proteins can be performed by avariety of methods known to those skilled in the art. In brief,immunoassays to measure the protein can be either competitive ornoncompetitive binding assays. In competitive binding assays, the sampleanalyte competes with a labeled analyte for specific binding sites on acapture agent bound to a solid surface. Preferably the capture agent isan antibody specifically reactive with CAIR proteins produced asdescribed above. The concentration of labeled analyte bound to thecapture agent is inversely proportional to the amount of free analytepresent in the sample.

In a competitive binding immunoassay, the CAIR protein present in thesample competes with labelled protein for binding to a specific bindingagent, for example, an antibody specifically reactive with the CAIRprotein. The binding agent may be bound to a solid surface to effectseparation of bound labelled protein from the unbound labelled protein.Alternately, the competitive binding assay may be conducted in liquidphase and any of a variety of techniques known in the art may be used toseparate the bound labelled protein from the unbound labelled protein.Following separation, the amount of bound labeled protein is determined.The amount of protein present in the sample is inversely proportional tothe amount of labelled protein binding.

Alternatively, a homogenous immunoassay may be performed in which aseparation step is not needed. In these immunoassays, the label on theprotein is altered by the binding of the protein to its specific bindingagent. This alteration in the labelled protein results in a decrease orincrease in the signal emitted by label, so that measurement of thelabel at the end of the immunoassay allows for detection or quantitationof the protein.

CAIR proteins may also be detected and quantified by a variety ofnoncompetitive immunoassay methods. For example, a two-site, solid phasesandwich immunoassay is used. In this type of assay, a binding agent forthe protein, for example an antibody, is attached to a solid phase. Asecond protein binding agent, which may also be an antibody, and whichbinds the protein at a different site, is labelled. After binding atboth sites on the protein has occurred, the unbound labelled bindingagent is removed and the amount of labelled binding agent bound to thesolid phase is measured. The amount of labelled binding agent bound isdirectly proportional to the amount of protein in the sample.Alternatively, CAIR proteins may be detected using immunoprecipitationmethods as described by Otto et al. pages 119 to 127 in Methods in CellBiology Volume 37: Antibodies in Cell Biology, Asai, ed., AcademicPress, New York (1993).

Western blot analysis can also be done to determine the presence of CAIRproteins in a sample. Electrophoresis is carried out, for example, on atissue sample suspected of containing the protein. Followingelectrophoresis to separate the proteins, and transfer of the proteinsto a suitable solid support such as a nitrocellulose filter, the solidsupport is then incubated with an antibody reactive with the protein.This antibody may be labelled, or alternatively may be it may bedetected by subsequent incubation with a second labelled antibody thatbinds the primary antibody.

The immunoassay formats described above employ labelled assaycomponents. The label can be in a variety of forms. The label may becoupled directly or indirectly to the desired component of the assayaccording to methods well known in the art. A wide variety of labels maybe used. The component may be labelled by any one of several methods.Traditionally a radioactive label incorporating ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C,or ³² P was used. Non-radioactive labels include ligands which bind tolabelled antibodies, fluorophores, chemiluminescent agents, enzymes, andantibodies which can serve as specific binding pair members for alabelled ligand. The choice of label depends on sensitivity required,ease of conjugation with the compound, stability requirements, andavailable instrumentation. For a review of various labeling or signalproducing systems which may be used, see U.S. Pat. No. 4,391,904, whichis incorporated herein by reference.

Antibodies reactive with a particular protein can also be measured by avariety of immunoassay methods. For a review of immunological andimmunoassay procedures applicable to the measurement of antibodies byimmunoassay techniques, see Basic and Clinical Immunology 7th Edition(D. Stites and A. Terr ed.) supra, Enzyme Immunoassay, E. T. Maggio,ed., supra, and Harlow and Lane, Antibodies, A Laboratory Manual, supra.

In brief, immunoassays to measure antisera reactive with CAIR proteinscan be either competitive or noncompetitive binding assays. Incompetitive binding assays, the sample analyte competes with a labeledanalyte for specific binding sites on a capture agent bound to a solidsurface. Preferably the capture agent is a purified recombinant CAIRprotein produced as described above. Other sources of CAIR proteins,including isolated or partially purified naturally occurring protein,may also be used. Noncompetitive assays are typically sandwich assays,in which the sample analyte is bound between two analyte-specificbinding reagents. One of the binding agents is used as a capture agentand is bound to a solid surface. The second binding agent is labelledand is used to measure or detect the resultant complex by visual orinstrument means. A number of combinations of capture agent and labelledbinding agent can be used. A variety of different immunoassay formats,separation techniques and labels can be also be used similar to thosedescribed above for the measurement of CAIR proteins.

This invention also embraces kits for detecting the presence of CAIRproteins in tissue or blood samples which comprise a containercontaining antibodies selectively immunoreactive to the protein andinstructional material for performing the test. The kit may also containother components such as CAIR proteins, controls, buffer solutions, andsecondary antibodies.

This invention further embraces kits for detecting DNA or RNA encodingCAIR proteins in tissue or blood samples which comprise nucleic probesas described herein and instructional material. The kit may also containadditional components such as labeled compounds, as described herein,for identification of duplexed nucleic acids.

H. Detecting CAIR Resistance

A common problem in the treatment of various cancers is the acquisitionof resistance by the tumor cells to the particular pharmaceutical towhich they are exposed. It is desirable during therapy to assay for theacquisition of resistance to a particular pharmaceutical in order togauge the efficacy of treatment or to determine when to halt or alterparticular treatment protocols.

As shown in Example 1, tumor cells are capable of developing resistanceto CAI. The development of this resistance involves up-regulation ofCAIR gene and consequent expression of CAIR proteins. Thus, bothincreased levels of CAIR mRNA and CAIR proteins provide convenientmarkers to detect the onset of CAI resistance.

This invention provides for assays to detect CAI resistance. Theseassays require detecting the increased expression of CAIR proteins inthe sample tissues relative to the same tissues prior to their exposureto CAI and its functional equivalents. In a preferred embodiment, theseassays require obtaining a biological sample (e.g. tumor biopsy or bloodsample) prior to commencing a therapeutic regimen. During treatment withCAI or functional equivalents, subsequent samples will be taken andassayed for CAIR production. Where CAIR production shows an increaserelative to the initial (pre-CAI) samples, one may infer the onset ofCAI resistance.

CAIR production may be assayed by detecting expression levels of CAIRmRNA or CAIR proteins. Means for detecting CAIR mRNA are described insection (F), while means for detection CAIR proteins are described insection (G). In a preferred embodiment, mRNA will be detected usinghybridization probes, while CAIR proteins will be detected usingimmunoassays.

I. Use of CAI Resistant Cells in Screening for Resistance Modulators

The CAI resistant cells of the present invention may be utilized as in abioassay to screen for compounds that reduce or fully obviate CAIresistance. Generally such an assay will comprise culturing CAIresistant cells in the presence of fixed or varying concentrations ofthe compounds to be screened and CAI or functionally equivalentmolecules. Where the screened compounds reduce CAI resistance, thegrowth rate of the cells will decrease or the cells may die. Means ofdetermining cell growth rate are well known to those of skill in the artand include, for example, measuring changes in the uptake of labeledmetabolic substrates (e.g. [³ H]-thymidine) or by cell counts.

This invention further embraces kits for screening compositions orcombinations of compositions for therapeutic efficacy in CAI resistantcells which comprise CAI resistant cells as described herein andinstructional material. The kit may also contain additional componentssuch culture media, labeled substrates for assessing metabolic activityof the resistant cells as described herein for the screening of CAIresistance modulators.

J. Pharmacological Compositions

The nucleic acid sequences encoding the CAIR proteins and the CAIRproteins themselves may be utilized to produce anti-sense, antibody, andsingle chain peptide pharmacological compositions. The nucleotidesequence encoding CAI may be used to produce anti-sense therapeutics.These are molecules that bind to mRNA transcribed from the CAIR gene andform heteroduplex molecules that prevent further translation and therebyinhibit the expression of CAIR proteins causing the cell to loose itsCAI resistance. Methods of producing anti-sense molecules are well knownto those of skill in the art. See, for example, Cohen, et al., U.S. Pat.Nos. 5,264,423 and 5,276,019; Miller, et al., U.S. Pat. Nos. 4,469,863,and 4,757,055; and Uhlmann, et al. Chem. Rev., 90: 543-584 (1990).

Alternatively, the CAIR protein or fragments thereof may be used as animmunogen to generate antibodies specific to the CAIR protein asdescribed above. The antibodies will bind to the CAIR protein therebypreventing the normal interaction of that protein with other cellularconstituents. Again, the cell will loose its CAI resistance. Methods ofusing antibodies to block normal protein function are also well known tothose of skill in the art. See, for example, El-Badry, et al., CellGrowth and Diff, 1: 325-331 (1990).

The CAIR proteins may be used to make non-functional mimics that competewith the normal CAIR proteins and thereby reduce or modulate the effectof CAIR proteins. Means of producing disfunctional mimetics include thesubstitution or chemical modification of critical amino acid residues.Substitution of amino acids may be accomplished directly during thechemical synthesis or CAIR proteins, or if the proteins arerecombinantly expressed by using methods of site directed mutagenesis toalter the nucleotide sequence encoding particular residues. Means ofproduction of peptide mimetics are well known to those of skill in theart. For example the drug BB94 is a peptide mimetic that inhibits theenzyme collagenase and is used as a metastisis inhibitor.

As described above in section (B), the CAIR-1 resistance proteincontains a number of SH3 domain binding sites. SH3 domains have beenimplicated in a number of cellular signaling pathways as well ascytoskeletal activity. The CAIR protein or fragments thereof carryingthe SH3 binding domains may be used to selectively bind and block SH3domains thereby inhibiting specific signal transduction pathways. Thisis useful in the treatment of certain diseases characterized by abnormalsignaling such as cancer, hyperproliferative states, correction ofcollagen vascularities (e.g. lupis and rheumatoid arthritis),nephropathies, neruopathies (e.g. ALF, Alzheimers disease), andmyopathies (e.g. cardiomyopathies, skeletalmyopathies).

The pharmaceutical compositions of this invention are intended forparenteral, topical, oral or local administration. Preferably, thepharmaceutical compositions are administered parenterally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration whichcomprise a solution of the agents described above dissolved or suspendedin an acceptable carrier, preferably an aqueous carrier. A variety ofaqueous carriers may be used, e.g., water, buffered water, 0.4% saline,0.3% glycine, hyaluronic acid and the like. These compositions may besterilized by conventional, well known sterilization techniques, or maybe sterile filtered. The resulting aqueous solutions may be packaged foruse as is, or lyophilized, the lyophilized preparation being combinedwith a sterile solution prior to administration. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient and more preferably at a concentration of 25%-75%.

For aerosol administration, the polypeptides, antibodies, or antisensemolecules are preferably supplied in finely divided form along with asurfactant and propellant. The surfactant must, of course, be nontoxic,and preferably soluble in the propellant. Representative of such agentsare the esters or partial esters of fatty acids containing from 6 to 22carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic,linoleic, linolenic, olesteric and oleic acids with an aliphaticpolyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixedor natural glycerides may be employed. A carrier can also be included,as desired, as with, e.g., lecithin for intranasal delivery.

In therapeutic applications, antisense molecules, antibodies, orpolypeptides of the invention are administered to a patient in an amountsufficient to block expression of CAIR proteins or to block particularsignal transduction pathways. An amount adequate to accomplish this isdefined as "therapeutically effective dose." Amounts effective for thisuse will depend on, e.g., the particular antisense molecule, antibody,or polypeptide, the manner of administration, the weight and generalstate of health of the patient, and the judgment of the prescribingphysician.

K. Gene Therapy Applications

A variety of human diseases may be treated by therapeutic approachesthat involve stably introducing a gene into a human cell such that thegene may be transcribed and the gene product may be produced in thecell. Diseases amenable to treatment by this approach include inheriteddiseases, particularly those diseases such as GSD Type 1a where thedefect is with a single gene. For discussions on the application of genetherapy towards the treatment of genetic as well as acquired diseasesSee Miller, A. D. (1992) Nature 357:455460, and Mulligan, R. C. (1993)Science 260:926-932, both incorporated herein by reference.

This invention contemplates the use of gene therapy to introduce CAIRgenes into healthy cells so that they are spared and permit the exposureof unhealthy, e.g. tumor cells, to higher doses of CAI while reducingsystemic toxicity. Alternatively, gene therapy may be used to introducegenes encoding CAI mimetics that compete with CAI and thereby reduce thelevel of CAI resistance of diseased cells rendering them moresusceptible to CAI therapy.

Delivery of the gene or genetic material into the cell is the firstcritical step in gene therapy treatment of disease. A variety of methodshave been used experimentally. Most research has focused on the use ofretroviral and adenoviral vectors for gene delivery into the cell.Retroviral vectors have the ability to stably integrate the transferredgene sequences into the chromosomal DNA of the target cell. Retroviralvectors are particularly attractive because they are very efficient instably transducing a high percentage of target cells. Accordingly mostof the approved gene therapy clinical protocols use retroviral vectors.See Miller, A. D., (1992) supra. Retroviral vectors are particularlyuseful for modifying cells because of the high efficiency with which theretroviral vectors transduce target cells and integrate into the targetcell genome. Additionally, the retroviruses harboring the retroviralvector are capable of infecting cells from a wide variety of tissues.

Retroviral vectors are produced by genetically manipulatingretroviruses. Retroviruses are called RNA viruses because the viralgenome is RNA. Upon infection, this genomic RNA is reverse transcribedinto a DNA copy which is integrated into the chromosomal DNA oftransduced cells with a high degree of stability and efficiency. Theintegrated DNA copy is referred to as a provirus and is inherited bydaughter cells as is any other gene. The wild type retroviral genome andthe proviral DNA have three genes: the gag, the pol and the env genes,which are flanked by two long terminal repeat (LTR) sequences. The gaggene encodes the internal structural (nucleocapsid) proteins; the polgene encodes the RNA directed DNA polymerase (reverse transcriptase);and the env gene encodes viral envelope glycoproteins. The 5' and 3'LTRs serve to promote transcription and polyadenylation of virion RNAs.Adjacent to the 5' LTR are sequences necessary for reverse transcriptionof the genome (the tRNA primer binding site) and for efficientencapsulation of viral RNA into particles (the Psi site). See Mulligan,R. C., In: Experimental Manipulation of Gene Expression, M. Inouye (ed),155-173 (1983); Mann, R., et al., Cell, 33:153-159 (1983); Cone, R. D.and R. C. Mulligan, Proc. Natl. Acad. Sci., U.S.A., 81:6349-6353 (1984).

The design of retroviral vectors is well known to one of skill in theart. See Singer, M. and Berg, P. supra. In brief, if the sequencesnecessary for encapsidation (or packaging of retroviral RNA intoinfectious virions) are missing from the viral genome, the result is acis acting defect which prevents encapsidation of genomic RNA. However,the resulting mutant is still capable of directing the synthesis of allvirion proteins. Retroviral genomes from which these sequences have beendeleted, as well as cell lines containing the mutant genome stablyintegrated into the chromosome are well known in the art and are used toconstruct retroviral vectors. Preparation of retroviral vectors andtheir uses are described in many publications including European PatentApplication EPA 0 178 220, U.S. Pat. No. 4,405,712, Gilboa,Biotechniques 4:504-512 (1986), Mann, et al., Cell 33:153-159 (1983),Cone and Mulligan, Proc. Natl. Acad. Sci. U.S.A. 81:6349-6353 (1984),Eglitis, M. A, et al. (1988) Biotechniques 6:608-614, Miller, A. D. etal. (1989) Biotechniques 7:981-990, Miller, A. D.(1992) Nature, supra,Mulligan, R. C. (1993), supra. and Gould, B. et al., and InternationalPatent Application No. WO 92/07943 entitled "Retroviral Vectors Usefulin Gene Therapy". The teachings of these patents and publications areincorporated herein by reference.

The retroviral vector particles are prepared by recombinantly insertingthe gene encoding CAIR proteins or non-functional CAIR protein mimeticsinto a retrovirus vector and packaging the vector with retroviral capsidproteins by use of a packaging cell line. The resultant retroviralvector particle is incapable of replication in the host cell and iscapable of integrating into the host cell genome as a proviral sequencecontaining the CAIR gene or CAIR non-functional mimetic. As a result,the patient is capable of producing normal CAIR and resisting theeffects of calcium influx inhibitors or alternatively, the production ofnon-functional CAIR mimetics prevents the onset of CAI resistance.

Packaging cell lines are used to prepare the retroviral vectorparticles. A packaging cell line is a genetically constructed mammaliantissue culture cell line that produces the necessary viral structuralproteins required for packaging, but which is incapable of producinginfectious virions. Retroviral vectors, on the other hand, lack thestructural genes but have the nucleic acid sequences necessary forpackaging. To prepare a packaging cell line, an infectious clone of adesired retrovirus, in which the packaging site has been deleted, isconstructed. Cells comprising this construct will express all structuralproteins but the introduced DNA will be incapable of being packaged.Alternatively, packaging cell lines can be produced by transforming acell line with one or more expression plasmids encoding the appropriatecore and envelope proteins. In these cells, the gag, pol, and env genescan be derived from the same or different retroviruses.

A number of packaging cell lines suitable for the present invention arealso available in the prior art. Examples of these cell lines includeCrip, GPE86, PA317 and PG13. See Miller et al., J. Virol. 65: 2220-2224(1991), which is incorporated herein by reference. Examples of otherpackaging cell lines are described in Cone and Mulligan, Proc. Natl.Acad. Sci. U.S.A., 81:6349-6353 (1984) and in Danos and Mulligan, Proc.Natl. Acad. Sci. U.S.A., 85: 6460-6464 (1988), Eglitis, et al. (1988)supra and Miller, (1990) supra, also all incorporated herein byreference.

Packaging cell lines capable of producing retroviral vector particleswith chimeric envelope proteins may be used. Alternatively, amphotropicor xenotropic envelope proteins, such as those produced by PA317 and GPXpackaging cell lines may be used to package the retroviral vectors.

The following examples are offered by way of illustration, not by way oflimitation.

EXAMPLES Example 1

Culture of CAI Resistant Cells

Carboxyamido-triazole (CAI) has been observed to inhibit malignantproliferation, invasion, and metastasis of cancer cells, suggesting therole of CAI, and related compounds, as potential cancer therapeutics. Ofconcern in the development and utilization of cancer therapeutics is thedevelopment of resistance by tumor cells to the particularpharmacological regimen to which they are exposed. Thus, it was desiredto develop, cells that express a resistance to CAI both as a modelsystem for examining mechanisms of CAI resistance and to provide anassay or screening system for treatment regimens that reduce oreliminate resistance to CAI and its analogues.

Low passage (passage 13) parental human melanoma cells (A2058), andhuman ovarian cancer cells (OVCAR3) were cultured in Dulbecco's ModifiedEagles medium (DMEM) supplemented with 10% fetal calf serum (FCS) andpen/strep (penicillin and streptomycin). Over a period of 24 to 30months, the cells were incubated with increasing concentrations of CAIstarting with 0.1 μM and reaching 45 μM. The CAI, obtained from theDevelopmental Therapeutics Program of the National Cancer Institute, wasdissolved in DMSO and aliquots were stored at -70° C. until used. Thediluted CAI was added directly into the media as required. Continuousselective pressure with CAI was maintained throughout culture and wasmaintained throughout all experiments.

It was found that A2058 cells chronically resistant to 10 μM (10R), 20μM (20R), 30 μM (30R), and 40 μM (40R) were obtained. No overtdifferences in morphology of the A2058 CAI resistant cells was observed.The growth rate of A2058 CAI resistant cells was not attenuated untilthe 40 μM treatment in which it decreased somewhat.

OVCAR3 cells chronically resistant to 10 μM and 20 μM CAI were alsoestablished. The OVCAR3 cells had slightly lower growth rates in the CAItreatments.

Example 2

Isolation of the CAIR-1 Gene

DNA encoding a protein that correlated with CAI resistance in A2058cells was isolated by subtraction hybridization according to the methoddescribed by Hampson, et al. Nucleic Acids Res., 20: 2899 (1992).

RNA was isolated from 20 μM resistant A2058 cells (A2058-20R) and usedas a template to synthesize cDNA using reverse transcriptase. Theresulting cDNA was purified from the residual RNA by alkaline hydrolysis(0.5 M NaOH, 15 minutes, 55° C.) followed by a Sephadex G50 spin column.The cDNA was quantitated using ³² P-dCTP tracer and 500 ng was used forhybridization in solution to 10 μg mRNA obtained from the parental (wildtype) cell line (A2058). Hybridization was performed for 20 hours at 68°C. in 10 μL final volume with final concentrations of 0.5 M NaCl, 25 mMHEPES buffer, pH 7.5, 5 mM EDTA, and 1% SDS.

After hybridization, the final product was diluted 5-fold with moleculargrade double distilled H₂ O and then ethanol precipitated (1/10 volume 3M NaOAc, pH 5.0, 2 vol 100% ethanol, dry ice for 20 minutes or overnightat -20° C.). The resulting pellet was gently washed with 80% ethanol,dried under vacuum, and dissolved in 50 μL buffer (25 mM Tris-HCl, pH7.0, 1 mM EDTA, 5% DMSO, 2 mM ascorbic acid) and incubated for 3 minutesat 68° C.

To chemically crosslink the mRNA:cDNA heteroduplex,2,5-diaziridynyl-3,4-benzoquinone (DZQ) was added to a finalconcentration of 200 μM and the reaction mixture was incubated at 45° C.for 20 minutes. The reaction was then ethanol precipitated as describedabove, washed and used for probing either Northern blots, genomic DNA,or cDNA libraries. The probe was labelled with ³² P-dCTP using standardrandom primer techniques as described by Maniatis, et al. MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NewYork (1982) which is incorporated herein by reference. The labelingreaction was run at room temperature for 20 minutes using Sequenase IIenzyme.

The subtracted probe was first hybridized against a Northern blot oftotal RNA from parental A2058 cells and CAI resistant A2058-20R cells.Increased expression of a number of transcripts was seen (FIG. 1, upperleft panel). Rehybridization of the blot with radiolabeled GAPDH as ahousekeeping gene confirmed the increased expression (FIG. 1, lower leftpanel).

A cDNA library was constructed from A2058-20R cells by Stratagene (LaJolla, Calif., U.S.A.). The library was probed using standard approaches(Id.) and the positive probes were plaque purified. Forty-one cloneswere selected and 35 were determined to be nonoverlapping by restrictiondigest. Fifteen were randomly selected for determination of theirexpression by Northern analysis. Inserts were purified by low meltingpoint agarose gel electrophoresis and then radiolabeled with standardrandom primer conditions (Amersham, Little Chalfont, Bucks, UnitedKingdom) and hybridized with a Northern of parental (wildtype) total RNAand RNA from A2058-20R cells. Of the clones screened, three (Table 1)showed increased expression in response to culture with CAI. Table 1shows, for each clone, the size of the transcript as determined from theNorthern blots (FIG. 1) and the size of the cDNA insert. The CAIR-1 cDNA(clone 21DBB) which showed 3.8 fold (range 2 fold to ˜4 fold onreplicate Northerns) increased expression compared to theglyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping genecontrol was selected for sequence analysis. The CAIR-1 cDNA (clone 21DBBinsert) was sequenced using standard dideoxy-sequencing (SequenaseIIkit) with 35S-dATP label. The cDNA sequence is shown in SEQ ID NO:1.

                  TABLE 1                                                         ______________________________________                                        Subtraction hybridization results.                                                               Transcript                                                                             cDNA (insert)                                       Clone Size Size                                                             ______________________________________                                        21DBB (CAIR-1) 2.8 kb   1.3 kb                                                  13BAA (CAIR-2) 4.2 kb 1.9 kb                                                  15CBB (CAIR-3) 4.5 kb 2.1 kb                                                ______________________________________                                    

Example 3

Characterization of CAIR-1

The CAIR-1 cDNA sequence was analyzed using GeneWorks and GeneBank andfound to be unique and containing an open reading frame, apolyadenylation signal and a polyA tail indicating that it is the 3' endof a mammalian gene. Translation of the DNA sequence yielded thecarboxyl terminus of a protein which is unique by GeneBank and otherdatabase comparisons. The CAIR-1 sequence contains a unique proline richsequence which fulfills the consensus definition for Src homology 3(SH3) binding proteins. The SH3 domains are common in signaling proteinsand have been shown to target to cytoskeleton, membrane and othersignaling proteins (Koch, et al., Science 252: 668-674 (1991). The SH3BP domain consensus sequence is: XPXXPPPψXP where positions 2,7, and 10are obligate P (proline), the X is any amino acid, and the ψ is ahydrophobic amino acid. There are 4 unique versions of the SH3 bindingprotein consensus sequences in carboxyl terminus of CAIR-1 protein(Table 2).

                  TABLE 2                                                         ______________________________________                                        SH3 binding protein domain consensus sequences. CAIR-1                          A through D were identified in the CAIR protein                               encoded by clone 21DBB.                                                         Protein Id  Consensus     SEQ ID NO:                                      ______________________________________                                                    XPXXPPPψXP                                                                              (SEQ ID NO: 3)                                        CAIR-1 A KPVSDKPPPP  (SEQ ID NO: 4)                                           CAIR-1 B VPPAPVPCPP  (SEQ ID NO: 5)                                           CAIR-1 C APVPCPPPSP  (SEQ ID NO: 6)                                           CAIR-1 D APAEATPPKP  (SEQ ID NO: 7)                                           3BP-1.sup.1: APTMPPPLPP  (SEQ ID NO: 8)                                       p85α1.sup.2: APPTPKPRPP  (SEQ ID NO: 9)                                 p85α2.sup.3: QPAPALPPKP  (SEQ ID NO: 10)                              ______________________________________                                         .sup.1 Ren, et al. Science 259: 1157-1161 (1993)                              .sup.2,3 Kapeller, et al. J. Biol. Chem., 21: 1927-1233 (1994)           

Example 4

Expression of CAIR Proteins in Tissues

Commercial Northerns consisting of poly-A RNAs from human heart, brain,placenta, lung, liver, skeletal muscle, kidney, and pancreas, spleen,thymus, prostate, testis, ovary, small intestine, colon, and peripheralblood leukocytes were obtained from Stratagene (La Jolla, Calif.,U.S.A.). The preparations were probed with the CAIR-1 probe.

The human tissue distribution of CAIR-1 demonstrated marked expressionin heart, skeletal muscle (both known to have important calcium-mediatedregulation) and also in placenta, lung, and liver. Less expression wasnoted in brain, kidney, and pancreas. The expression in prostate,testis, ovary, and colon was minimal.

Example 5

Detection of SH3 Binding Regions in CAIR-1

The cDNA of CAIR-1 containing an SH3 binding domain is subcloned intopGEX-2T vectors at BamHI and EcoRI sites. Escherichia coli cells aretransformed with the subcloned pGEX2T vector. Expression of the CAIR-1fusion protein is induced with isopropyl-1-thio-B-D-galactopyranoside.The cells are then lysed and the lysate containing the GST-CAIR-1 fusionproteins are purified using glutathione agarose beads and eluted withTris-HCl containing reduced glutathione.

The purified GST-CAIR-1 fusion protein is immobilized onglutathione-Sepharose beads. The beads are then incubated at 4° C.(while rocking constantly) with the proteins containing SH3 domains suchas Abl, Fyn, Lck and p85. The beads are washed and the eluted proteinsare checked using SDS-PAGE, at 4° C. with constant rocking.

The SH3-CAIR-1 complex is then purified by the use ofglutathione-Sepharose beads and then electrophoretically separated. SH3binding complexes can be identified using commercially available SH3antibodies (antibody to the SH3-containing protein) or by antibody toCAIR-1.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 10                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1269 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..1269                                                         (D) OTHER INFORMATION: - #/standard.sub.-- name= "CAIR-1          cDNA"                                                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GGCACGAGTT CAAGTGATCC GCAAAGAGGT GGATTCTAAA CCTGTTTCCC AG -            #AAGCCCCC     60                                                                 - - ACCTCCCTCT GAGAAGGTAG AGGTGAAAGT TCCCCCTGCT CCAGTTCCTT GT -            #CCTCCTCC    120                                                                 - - CAGCCCTGGC CCTTCTGCTG TCCCCTCTTC CCCCAAGAGT GTGGCTACAG AA -            #GAGAGGGC    180                                                                 - - AGCCCCCAGC ACTGCCCCTG CAGAAGCTAC ACCTCCAAAA CCAGGAGAAG CC -            #GAGGCTCC    240                                                                 - - CCCAAAACAT CCAGGAGTGC TGAAAGTGGA AGCCATCCTG GAGAAGGTGC AG -            #GGGCTGGA    300                                                                 - - GCAGGCTGTA GACAACTTTG AAGGCAAGAA GACTGACAAA AAGTACCTGA TG -            #ATCGAAGA    360                                                                 - - GTATTTGACC AAAGAGCTGC TGGCCCTGGA TTCAGTGGAC CCCGAGGGAC GA -            #GCCGATGT    420                                                                 - - GCGTCAGGCC AGGAGAGACG GTGTCAGGAA GGTTCAGACC ATCTTGGAAA AA -            #CTTGAACA    480                                                                 - - GAAAGCCATT GATGTCCCAG GTCAAGTCCA GGTCTATGAA CTCCAGCCCA GC -            #AACCTTGA    540                                                                 - - AGCAGATCAG CCACTGCAGG CAATCATGGA GATGGGTGCC GTGGCAGCAG AC -            #AAGGGCAA    600                                                                 - - GAAAAATGCT GGAAATGCAG AAGATCCCCA CACAGAAACC CAGCAGCCAG AA -            #GCCACAGC    660                                                                 - - AGCAGCGACT TCAAACCCCA GCAGCATGAC AGACACCCCT GGTAACCCAG CA -            #GCACCGTA    720                                                                 - - GCCTCTGCCC TGTAAAAATC AGACTCGGAA CCGATGTGTG CTTTAGGGAA TT -            #TTAAGTTG    780                                                                 - - CATGCATTTC AGAGACTTTA AGTCAGTTGG TTTTTATTAG CTGCTTGGTA TG -            #CAGTAACT    840                                                                 - - TGGGTGGAGG CAAAACACTA ATAAAAGGGC TAAAAAGGAA AATGATGCTT TT -            #CTTCTATA    900                                                                 - - TTCTTACTCT GTACAAATAA AGAAGTTGCT TGTTGTTTCA GAAGTTTAAC CC -            #CGTTGCTT    960                                                                 - - GTTCTGCAGC CCTGTCTACT TGGGCACCCC CACCACCTGT TAGCTGTGGT TG -            #TGCACTGT   1020                                                                 - - CTTTTGTAGC TCTGGACTGG AGGGGTAGAT GGGGAGTCAA TTACCCATCA CA -            #TAAATATG   1080                                                                 - - AAACATTTAT CAGAAATGTT GCCATTTTAA TGAGATGATT TTCTTCATCT CA -            #TAATTAAA   1140                                                                 - - ATACCTGACT TTAGAGAGAG TAAAATGTGC CAGGAGCCAT AGGAATATCT GT -            #ATGTTGGA   1200                                                                 - - TGACTTTAAT GCTACATTTT AAAAAAAGAA AATAAAGTAA TAATATAACT CA -            #AAAAAAAA   1260                                                                 - - AAAAAAAAA                - #                  - #                      - #       1269                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 241 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..241                                                          (D) OTHER INFORMATION: - #/note= "CAIR-1 protein"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Asn Ser Ala Arg Val Gln Val Ile Arg Lys Gl - #u Val Asp Ser Lys Pro      1               5   - #                10  - #                15               - - Val Ser Gln Lys Pro Pro Pro Pro Ser Glu Ly - #s Val Glu Val Lys Val                  20      - #            25      - #            30                   - - Pro Pro Ala Pro Val Pro Cys Pro Pro Pro Se - #r Pro Gly Pro Ser Ala              35          - #        40          - #        45                       - - Val Pro Ser Ser Pro Lys Ser Val Ala Thr Gl - #u Glu Arg Ala Ala Pro          50              - #    55              - #    60                           - - Ser Thr Ala Pro Ala Glu Ala Thr Pro Pro Ly - #s Pro Gly Glu Ala Glu      65                  - #70                  - #75                  - #80        - - Ala Pro Pro Lys His Pro Gly Val Leu Lys Va - #l Glu Ala Ile Leu Glu                      85  - #                90  - #                95               - - Lys Val Gln Gly Leu Glu Gln Ala Val Asp As - #n Phe Glu Gly Lys Lys                  100      - #           105      - #           110                  - - Thr Asp Lys Lys Tyr Leu Met Ile Glu Glu Ty - #r Leu Thr Lys Glu Leu              115          - #       120          - #       125                      - - Leu Ala Leu Asp Ser Val Asp Pro Glu Gly Ar - #g Ala Asp Val Arg Gln          130              - #   135              - #   140                          - - Ala Arg Arg Asp Gly Val Arg Lys Val Gln Th - #r Ile Leu Glu Lys Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Glu Gln Lys Ala Ile Asp Val Pro Gly Gln Va - #l Gln Val Tyr Glu        Leu                                                                                             165  - #               170  - #               175             - - Gln Pro Ser Asn Leu Glu Ala Asp Gln Pro Le - #u Gln Ala Ile Met Glu                  180      - #           185      - #           190                  - - Met Gly Ala Val Ala Ala Asp Lys Gly Lys Ly - #s Asn Ala Gly Asn Ala              195          - #       200          - #       205                      - - Glu Asp Pro His Thr Glu Thr Gln Gln Pro Gl - #u Ala Thr Ala Ala Ala          210              - #   215              - #   220                          - - Thr Ser Asn Pro Ser Ser Met Thr Asp Thr Pr - #o Gly Asn Pro Ala Ala      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Pro                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #/note= "X = any amino acid"               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "X = any amino acid"                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "X = any amino acid"                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                               (D) OTHER INFORMATION: - #/note= "X = a hydrophobic amino          acid"                                                                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                               (D) OTHER INFORMATION: - #/note= "X = any amino acid"               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Xaa Pro Xaa Xaa Pro Pro Pro Xaa Xaa Pro                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Lys Pro Val Ser Asp Lys Pro Pro Pro Pro                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Val Pro Pro Ala Pro Val Pro Cys Pro Pro                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Ala Pro Val Pro Cys Pro Pro Pro Ser Pro                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Ala Pro Ala Glu Ala Thr Pro Pro Lys Pro                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Ala Pro Thr Met Pro Pro Pro Leu Pro Pro                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Ala Pro Pro Thr Pro Lys Pro Arg Pro Pro                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Gln Pro Ala Pro Ala Leu Pro Pro Lys Pro                                  1               5   - #                10                                    __________________________________________________________________________

What is claimed is:
 1. An isolated antibody that specifically binds aCAIR protein comprising a polypeptide having an amino acid sequence ofSEQ ID NO:
 2. 2. The antibody of claim 1, wherein said antibody isgenerated against an immunogen consisting of the amino acid sequencedepicted by SEQ ID NO:2.
 3. The antibody of claim 1, wherein saidantibody is a monoclonal antibody.
 4. A kit comprising a containercontaining an isolated antibody that specifically binds a CAIR proteincomprising a polypeptide having an amino acid sequence of SEQ ID NO: 2.5. The kit of claim 4, wherein said antibody is generated against animmunogen consisting of the amino acid sequence depicted by SEQ ID NO:2.6. The kit of claim 4, wherein said antibody is a monoclonal antibody.